Moss Tom. DNA-protein interactions: principles and protocols

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Edited by

Tom Moss

Methods in Molecular Biology

VOLUME 148

DNA–Protein

Interactions

SECOND

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SECOND

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Principles and Protocols

Edited by

Tom Moss

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DNA-protein interactions : principles and protocols / edited by Tom Moss.--2nd ed.

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Includes bibliographical references and index.

ISBN 0-89603-625-1 (hc : alk. paper) -- ISBN 0-89603-671-5 (pbk.: alk. paper)

1. DNA-protein interactions. I. Moss, Tom. II. Series.

QP624.75.P74 D57 2001

572.8'6--dc21 00-054100

CIP

DNA–protein interactions are fundamental to the existence of life forms,

providing the key to the genetic plan as well as mechanisms for its mainte-

nance and evolution. The study of these interactions is therefore fundamental

to our understanding of growth, development, differentiation, evolution, and

disease. The manipulation of DNA–protein interactions is also becoming increas-

ingly important to the biotechnology industry, permitting among other things

the reprogramming of gene expression. The success of the first edition of DNA–

Protein Interactions; Principles and Protocols was the result of Dr. G. Geoff

Kneale's efforts in bringing together a broad range of relevant techniques. In

producing the second edition of this book, I have tried to further increase this

diversity while presenting the reader with alternative approaches to obtaining

the same information.

A major barrier to the study of interactions between biological macro-

molecules has always been detection and hence the need to obtain sufficient

material. The development of molecular cloning and subsequently of protein

overexpression systems has essentially breached this barrier. However, in the

case of DNA–protein interactions, the problem of quantity and hence of de-

tection is often offset by the high degree of selectivity and stability of DNA–

protein interactions. DNA–protein binding reactions will often go to near

completion at very low component concentrations even within crude protein

extracts. Thus, although many techniques described in this volume were ini-

tially developed to study interactions between highly purified components,

these same techniques are often just as applicable to the identification of novel

DNA–protein interactions within systems as undefined as a whole cell extract

In general, these techniques use a DNA rather than a protein detection system

because the former is more sensitive. Radiolabeled DNA fragments are easily

produced by a range of techniques commonly available to molecular biolo-

gists.

DNA–protein complexes may be studied at three distinct levels—at the

level of the DNA, of the protein, and of the complex. At the level of the DNA,

the DNA binding site may be delimited and exact base sequence requirements

defined. The DNA conformation can be studied and the exact bases contacted

Preface

vi Preface

by the protein identified. At the protein level, the protein species binding a

given DNA sequence can be identified. The amino acids contacting DNA and

the protein surface facing the DNA may be defined and the amino acids essential

to the recognition process can be identified. Furthermore, the protein’s tertiary

structure and its conformational changes on complex formation can be stud-

ied. Finally, global parameters of a DNA–protein complex such as stoichiom-

etry, the kinetics of its formation and dissociation, its stability, and the energy

of interaction can be measured.

Filter binding, electrophoretic mobility shift assay (EMSA/gel shift),

DNaseI footprinting, and Southwestern blotting have been the most commonly

used techniques to identify potentially interesting DNA target sites and to define

the proteins that bind them. For example, gel shift or footprinting of a cloned

gene regulation sequence by proteins in a crude cell extract may define binding

activities for a given DNA sequence that correlates with gene expression or

silencing. These techniques can be used as an assay during subsequent isolation

of the protein(s) responsible. Interference assays, SELEX, and more refined

footprinting techniques, such as hydroxy radical footprinting and DNA bend-

ing assays, can then be used to study the DNA component of the DNA–protein

complex, whereas the protein binding surface can be probed by amino acid

side chain modification, DNA–protein crosslinking, and of course by the pro-

duction of protein mutants. Genetic approaches have also opened the way to

engineer proteins recognizing chosen DNA targets.

DNA–protein crosslinking has in recent years become a very important

approach to investigate the relative positions of proteins in multicomponent

protein–DNA complexes such as the transcription initiation complex. Here,

crosslinkable groups are incorporated at specific DNA sequences and these

are used to map out the “positions” of different protein components along the

DNA. Extension of this technique can also allow the mapping of the crosslink

within the protein sequence. Similar data can be obtained by incorporating

crosslinking groups at known sites within the protein and then identifying the

nucleotides targeted.

Once the basic parameters of a DNA–protein interaction have been

defined,

it is inevitable that a deeper understanding of the driving forces

behind the DNA–protein interaction and the biological consequences of its

formation will require physical and physicochemical approaches. These can

be either static or dynamic measurements, but most techniques have been

developed to deal with steady-state situations. Equilibrium constants can be

obtained by surface plasmon resonance, by spectroscopic assays that differen-

tiate complexed and uncomplexed components, and, for more stable products,

by footprinting and gel shift. Spectroscopy can also give specific answers about

Preface vii

the conformation of proteins and any conformational changes they undergo

on interacting with DNA as well as providing a rapid quantitative measure of

complex formation. Microcalorimetry gives a global estimation of the forces

stabilizing a given complex. Static pictures of protein–DNA interactions can be

obtained by several techniques. At atomic resolution, X-ray crystallography,

and nuclear magnetic resonance (NMR) studies require large amounts of highly

homogeneous material. Lower resolution images can be obtained by electron

and, more recently, by atomic force microscopies. Large multiprotein com-

plexes are generally beyond the scope of NMR or even of X-ray crystallogra-

phy. These are therefore more often studied using the electron microscope,

either in a direct imaging mode or via the analysis of data obtained from 2D

pseudocrystalline arrays.

Dynamic measurements of complex formation or dissociation can be

obtained by biochemical techniques when the DNA–protein complexes have

half-lives of several minutes to several hours. For footprinting and crosslinking,

a general rule is that the complexes should be stable for a time well in excess

of the proposed period of the enzymatic or chemical reaction. For gel shift, the

complex half-life should at least approach that of the time of gel migration,

although the cage effect may tend to stabilize the complex within the gel ma-

trix, extending the applicability of this technique. More rapid assembly kinet-

ics, multistep assembly processes, and short-lived DNA–protein complexes

require much more rapid techniques such as UV laser-induced crosslinking,

surface plasmon resonance, and spectroscopic assays. UV-laser induced DNA–

protein crosslinking is a promising development because it potentially per-

mits the kinetics of complex assembly to be followed both in vitro and in vivo.

When I decided to edit a second edition of the present volume, I was of

course aware of the limitations of many of the more commonly used tech-

niques. But as I read the various chapters I realized that each technique was at

least as much limited by the conditions necessary for the probing reaction

itself as by the type of information the probe could deliver. This is perhaps

most evident for in vivo applications, which require agents that can easily

enter cells, e.g., DMS and potassium permanganate are able to penetrate cells

while DNaseI and DEPC are either too large or insufficiently water soluble to

enter cells unaided. (Appendix II presents a summary of the activities and

applications of the various DNA modification and cleavage reagents described

in this book.) Gel shift assays are limited by the finite range of useable elec-

trophoresis conditions. Because buffers must have low conductance, the KCl

or NaCl solutions typically used for DNA–protein binding reactions are gen-

erally inappropriate. (Appendix I contains a list of the different gel shift

conditions described in various chapters of this book.) Thus, it is often as

viii Preface

important to choose a technique appropriate to the conditions under which

one wishes to observe the DNA–protein interaction as it is to choose the

appropriate probing activity.

The present volume attempts to bring together a broad range of tech-

niques used to study DNA–protein interactions. Such a volume can never be

complete nor definitive, but I hope this book will provide a useful source of

technical advice for molecular biologists. Its preparation required the coop-

eration of many people. In particular I would like to thank all the authors for

their very significant efforts. Thanks are also due to John Walker for his

encouragement and to the previous editor Geoff Kneale and to Craig Adams

of Humana Press for their help. I also thank Margrit and Peter Wittwer for

providing space in the Pfarrhaus of the Predigerkirche, Zürich, where much of

the chapter editing was done, and Bernadette for her patience, understanding,

corrections, and advice.

Tom Moss

Contents

Preface .............................................................................................................

Contributors ...................................................................................................

xiii

1Filter-Binding Assays

Peter G. Stockley ................................................................................... 1

2Electrophoretic Mobility Shift Assays for the Analysis

of DNA–Protein Interactions

Marc-André Laniel, Alain Béliveau, and Sylvain L. Guérin ............ 13

3 DNase I Footprinting

Benoît Leblanc and Tom Moss .......................................................... 31

4Footprinting with Exonuclease III

Willi Metzger and Hermann Heumann............................................... 39

5Hydroxyl Radical Footprinting

Evgeny Zaychikov, Peter Schickor, Ludmilla Denissova,

and Hermann Heumann .................................................................. 49

6The Use of Diethyl Pyrocarbonate and Potassium

Permanganate as Probes for Strand Separation and Structural

Distortions in DNA

Brenda F. Kahl and Marvin R. Paule ................................................. 63

7Footprinting DNA–Protein Interactions in Native Polyacrylamide Gels

by Chemical Nucleolytic Activity of 1,10-Phenanthroline-Copper

Athanasios G. Papavassiliou ............................................................. 77

8Uranyl Photofootprinting

Peter E. Nielsen.................................................................................. 111

9Osmium Tetroxide Modification and the Study

of DNA–Protein Interactions

James A. McClellan ........................................................................... 121

10 Determination of a Transcription-Factor-Binding Site by Nuclease

Protection Footprinting onto Southwestern Blots

Athanasios G. Papavassiliou ........................................................... 135

11 Diffusible Singlet Oxygen as a Probe of DNA Deformation

Malcolm Buckle and Andrew A. Travers ........................................ 151

xContents

12 Ultraviolet-Laser Footprinting

Johannes Geiselmann and Frederic Boccard ............................... 161

13 In Vivo DNA Analysis

Régen Drouin, Jean-Philippe Therrien, Martin Angers,

and Stéphane Ouellet.................................................................... 175

14 Identification of Protein–DNA Contacts with Dimethyl Sulfate:

Methylation Protection and Methylation Interference

Peter E. Shaw and A. Francis Stewart ............................................ 221

15 Ethylation Interference

Iain W. Manfield and Peter G. Stockley........................................... 229

16 Hydroxyl Radical Interference

Peter Schickor, Evgeny Zaychikov, and Hermann Heumann ...... 245

17 Identification of Sequence-Specific DNA-Binding Proteins

by Southwestern Blotting

Simon Labbé, Gale Stewart, Olivier LaRochelle,

Guy G. Poirier, and Carl Séguin .................................................. 255

18 A Competition Assay for DNA Binding Using the Fluorescent

Probe ANS

Ian A. Taylor and G. Geoff Kneale ................................................... 265

19 Site-Directed Cleavage of DNA by Linker Histone Protein-Fe(II)

EDTA Conjugates

David R. Chafin and Jeffrey J. Hayes ............................................. 275

20 Nitration of Tyrosine Residues in Protein–Nucleic Acid Complexes

Simon E. Plyte .................................................................................... 291

21 Chemical Modification of Lysine by Reductive Methylation:

A Probe of Residues Involved in DNA Binding

Ian A. Taylor and Michelle Webb ..................................................... 301

22 Limited Proteolysis of Protein–Nucleic Acid Complexes

Simon E. Plyte and G. Geoff Kneale................................................ 315

23 Ultraviolet Crosslinking of DNA–Protein Complexes

via 8-Azidoadenine

Rainer Meffert, Klaus Dose, Gabriele Rathgeber,

and Hans-Jochen Schäfer ............................................................ 323

24 Site-Specific Protein–DNA Photocrosslinking:

Analysis of Bacterial

Transcription Initiation Complexes

Nikolai Naryshkin, Younggyu Kim, Qianping Dong,

and Richard H. Ebright ................................................................. 337